Recent studies suggest that conventional laboratory isolates of HIV-1 may differ from the real virus challenge that infects humans. In order to be protective, antibodies must neutralize these transmitted viruses, which are variously described as fresh isolates or macrophage tropic isolates. We have begun to study the infectivity of such isolates, in order to understand what type of antibodies will be needed for protection. Our viruses come from two sources, reflecting two routes of infection. One is a classical macrophage tropic virus, BAL, obtained from lung washings. The other source is virus transmitted to babies. These viruses will not grow on the CD4 positive HeLa cell monolayer, which we have used in our earlier studies of T cell tropic virus. But they will grow on a new cell line, SW480, which was transfected with the genes for host CD4 and viral tat protein and which supports the growth of macrophage tropic virus. We are trying to develop a plaque-forming assay, in order to measure viral infectivity. In general, these plaques are highly sensitive to neutralizing antibodies, because each plaque is the result of a single viral infection event. This would allow us to measure the neutralizing antibodies present in a mother at the time of birth, and to determine the effect of these antibodies on viral transmission. It has been found that viral burden predicts risk of transmission, and this could be a result of neutralizing antibodies. In addition, the baby's virus usually turns out to be a single example of what is in the mother. We will determine whether the maternal neutralizing antibodies fail to neutralize this isolate, or whether transmission is simply a rare event, and the isolate transmitted is no more resistant than the ones that fail to transmit infection to the baby. We will also map neutralizing epitopes. This will help greatly in vaccine design. The T cell tropic viruses were shown to have a broadly crossreacting neutralizing site at the CD4 binding site that depended on the native protein conformation. This placed a number of constraints on any vaccine. Similarly, macrophage tropic virus may have its own neutralizing sites, each with their own conformational requirements. This information is critical to the design of vaccines against transmitted virus.